The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) is essential for normal central nervous system (CNS) function. However, excessive activation of NMDARs, particularly of extrasynaptic as opposed to synaptic receptors, mediates, at least in part, neuronal or synaptic damage in many neurological disorders. The dual role of NMDARs in normal and abnormal CNS function imposes important constraints on possible therapeutic strategies aimed at ameliorating or abating developmental disorders and neurological disease: blockade of excessive NMDAR activity must be achieved without interference with its normal function. One approach we propose is to utilize the inhibitory effect of a novel family of NMDAR subunits, the NR3A and NR3B, to downregulate excessive activity of NMDARs by affecting channel permeability. NR3 subunits have a unique structure in the channel region, which contributes to decreased magnesium sensitivity and calcium permeability. Additionally, glycine binding to the ligand-binding domain (LBD) of NR3 is essential for NR1/NR3 receptor activation, suggesting a critical role of the NR3 LBD in activation of NR3-containing heteromeric receptors. Also, compared to NR1, the NR3 LBD manifests differential sensitivity to NR1 agonists and antagonists, suggesting a unique structure of the NR3 LBD. However, currently there is no NR3-selective agonist, antagonist, or modulator. Therefore, we will utilize structural/functional properties of the NR3 LBD to design screening assays to discover chemical probes that selectively modulate NR3-containing receptors. NR3-containing NMDARs in neurons and myelin may be involved in more neurological diseases than previously thought, especially in so called white matter diseases, including multiple sclerosis, cerebral palsy (periventricular leukomalacia), and spinal cord injury. These new NR3 chemical probes identified in our screening efforts will not only be useful for further characterization of NR3-containing receptors in vitro and in vivo, but also may prove useful as drugs for neuroprotection. Accordingly, the Specific Aims of this proposal are as follows: Specific Aim 1. Implement an HTS assay for the NR3-LBD and screen a large-scale compound collection for identification of NR3 modulators. Specific Aim 2. Perform secondary assays for identification of hits that selectively bind to NR3 subunits and modulate NR3-containing receptors. Specific Aim 3. Establish and utilize structure-activity relationship (SAR) for optimization of the hits that modulate NR3-containing receptors.